The overall goal of this proposed research is to determine the functional significance of chemical heterogeneity in nucleosomes. Chemically different nucleosomes exist because different accessory proteins (Hl and non-histones) are associated with the DNA:histone octamer complex. In addition, histone octamers are heterogeneous because of the presence of histone variants (and post-translational modifications). Finally, DNA packaged around histone octamers is heterogeneous not only in nucleotide sequence but in its content of 5-methyl-cytosine (m5Cyt). We therefore wish to ask the following specific questions: 1. Is histone Hl non-randomly associated with nucleosomes in a functionally significant way? That is, do nucleosomes which reside within a specific transcription unit lack histone Hl when the transcription unit is active, but possess histone Hl when the transcription unit is inactive? 2. Are nonhistone chromosonal proteins HMG-14 and 17 non-randomly associated with nucleosomes in a functionally significant way? That is, do nucleosomes which reside within a specific transcription unit possess specific non-histone proteins when the transcription unit is active, but lack these proteins when the transcription unit is inactive? 3. Are core histone variants associated with a non-random population of nuclear DAN sequences? 4. Are DNA sequences which are enriched in m5Cyt localized in nucleosome classes which possess specific proteins? Our general experimental strategy toward the solution of these questions is first to fractionate nucleosomes based on their protein composition (using gel electrophoretic and immunological techniques), and then to study the distributions of either specific DNA sequences (primarily using cloned sequences as hybridization probes) or m5Cyt (using an antibody probe). Specific biological systems and genes have been chosen for these studies.